Differential pathogenetic mechanisms of mutations in helix 2 and helix 6 of rhodopsin

Variants in rhodopsin (RHO) have been linked to autosomal dominant congenital stationary night blindness (adCSNB), which affects the ability to see in dim light, and the pathogenetic mechanism is still not well understood. In this study we report two novel RHO variants found in adCSNB families, p.W265R and p.A269V, that map in the sixth transmembrane domain of RHO protein. We applied in silico molecular simulation and in vitro biochemical and molecular studies to characterize the two new variants and compare the molecular determinants to two previously characterized adCSNB variants, p.G90D and p.T94I, that map in the second transmembrane domain of the RHO protein. We demonstrate that W265R and A269V cause constitutive activation of RHO with light-independent G protein coupling and impaired binding to arrestin. Differently, G90D and T94I are characterized by slow kinetics of RHO activation and deactivation. This study provides new evidence on the differential contribution of transmembrane α-helixes two and six to the interaction with intracellular transducers of RHO and mutations in these helixes result in a similar phenotype in patients but with distinct molecular effects.